title: Fl3
description: Protocols: The BY4741 (MATa ura3 his3 leu2 met15 TRP1) was used as a parental strain in which the promoter of Npl3 was replaced with an inducible gal promoter by homologues recombination. The strain was complemented with pRS315::Npl3500Kb::HRV-3C::CterHTP plasmid. In this plasmid, the expression of Npl3 is driven by its endogenous promoter. A HRV-C3 protease cleavage site was inserted between amino acids S282 and N283 using PCR. A His-Trypsin-Protein A (HTP) tandem tag was placed at the C-terminus of the protein by PCR. The split-iCRAC was based on the original CRAC protocol described by Granneman et al. 2009 with some modifications (Granneman, Kudla et al., 2009). Briefly, the recombinant yeast strains were grown in SD-leu medium to drive Npl3 expression only from the transformed plasmid. 2 L of yeast culture were harvested at an OD600 of ~2. The cells were resuspended in 1 v/w SD-Trp medium and UV-irradiated (1.6 J/cm2) in Petri dishes in a Stratalinker 1600 (Stratagene). Half the cells were not subjected to UV treatment and were kept as the UV minus control. Cells were pelleted and resuspended in 25 ml of lysis buffer (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 0.15% NP-40) with addition of 1.3 mM PMSF, 1 mM DDT, complete protease inhibitor cocktail (Roche)). The cells were lysed using 25 ml glass beads in Planetary mill for 20 mins at 500 rpm. 5 ml of lysis buffer was added, and the lysate was cleared by centrifugation (20 min at 5000 RPM and 4 °C followed by 2x 20 min at 18000 RPM and 4 °C). Cleared lysate was incubated with 300 of IgG bead suspension (1:1) for 2 h at 4 °C with rotation. Beads were collected and washed 3x with 10 ml wash buffer (50 mM Tris-HCl pH 7.8, 1 M NaCl, 0.15% NP-40, 0.5 mM DDT) followed by 3x with 10 ml lysis buffer with added 0.5 mM DDT. Beads were resuspended in 5 ml of lysis buffer and rotated with 40 g of homemade TEV for 18 h at 4 °C. The eluates were concentrated to a volume of 500 l in 30 kDa cutoff centricons (Millipore). No RNase treatment was performed in the final experiments for library preparation. 0.4 g of Guanidine-HCl were dissolved in the eluate to yield a final concentration of 6M. NaCl and imidazole were added to a final concentration of 300 and 10 mM, respectively. 100 l of pre-equilibrated Ni-beads were added to the samples and incubated for 2h at 4 °C. Beads were washed 3x with wash buffer (6 M guanidine-HCl, 50 mM Tris-HCl (pH 7.8), 300 mM NaCl, 0.1% NP-40, 40 mM imidazole, and 1 mM DDT) followed by 3x with PNK buffer (50 mM Tris-HCl (pH 7.8), 40 mM MgCl2, 0.1% NP-40, and 1 mM DDT). The beads were then incubated in 50 l of PNK buffer (pH 6.5) in the presence of 10 units of PNK (NEB) and 10 units of CIP (NEB) and 20 units of SUPERase.In (Ambion) for 30 mins at 37 °C. Beads were washed 3x with wash buffer followed by 2x with PNK buffer (pH 7.8) and incubated with 50 pmoles of L3 adapter (rAppAGATCGGAAGAGCGGRRCAG/ddC/) in 50 l of ligation mixture (1x PNK buffer (pH 7.8) supplemented with 12.5 units of T4 RNA ligase 1 (NEB), 250 units of T4 RNA ligase 2 truncated K227Q (NEB), 40 units of SUPERase.In (Ambion) and 10% PEG 4000). The reaction is incubated for 18 h with mild shaking at 16 °C. Beads are then washed with 3x wash buffer followed by 3x PNK buffer. For the PreScission cleaved version, the beads are then incubated with 1x PNK buffer (pH 7.8) supplemented with 20 units of SUPERase.In (Ambion) and 10 units of PreScission protease (GE Healthcare) for 18 h at 4 °C with mild shaking. The mixture is then supplemented with 10 units of PNK and 0.5 l of 32P-ATP and incubated for 1 h at 37 °C. 20 l of 4x LDS is added to the beads, boiled for 10 mins and resolved on a 4-12% Bis-Tris NuPage gel (Invitrogen). Subsequent steps of RNA isolation and library preparation were done as described in Huppertz et al. 2014 Methods (Huppertz et al., 2014). The negative control sample was generated following the same procedure but starting with WT yeast strain that does not express any tagged protein.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-09-01
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-09-01
attributes.tag: External Id
attributes.value: SAMEA14382049
attributes.tag: INSDC center alias
attributes.value: UZH
attributes.tag: INSDC center name
attributes.value: University of Zurich
attributes.tag: INSDC first public
attributes.value: 2022-09-01T00:14:15Z
attributes.tag: INSDC last update
attributes.value: 2022-09-01T00:14:15Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11736:Fl3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: baker's yeast
attributes.tag: genotype
attributes.value: R64-1-1.83
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-11736:Fl3
attributes.tag: strain
attributes.value: BY4741